Bone is a complex mineralizing system composed of an inorganic or mineral phase, an organic matrix phase, and water. The inorganic mineral phase is composed mainly of crystalline calcium phosphate salts while the organic matrix phase consists mostly of collagen and other noncollagenous proteins. Calcification of bone depends on the close association between the organic and inorganic phases to produce a mineralized tissue.
The process of bone growth is regulated to meet both structural and functional requirements. The cells involved in the processes of bone formation, maintenance, and resorption are osteoblasts, osteocytes, and osteoclasts. Osteoblasts synthesize the organic matrix, osteoid, of bone which after calcium phosphate crystal growth and collagen assembly becomes mineralized. Osteocytes regulate the flux of calcium and phosphate between the bone mineral and the extracellular fluid. Osteoclasts function to resorb bone and are essential in the process of bone remodeling. Disturbing the natural balance of bone formation and resorption leads to various bone disorders. Increased osteoclast activity has been demonstrated to lead to bone disease characterized by a decrease in bone density such as that seen in osteoporosis, osteitis fibrosa and in Paget's disease. All of these diseases are a result of increased bone resorption.
In order to understand the mechanisms involved which regulate bone cell function, it is important to be able to assess the normal function of bone cells and also the degree of perturbation of this activity in various bone diseases. This will lead to the identification of drugs targeted to restore abnormal bone cell activity back to within normal levels. Together with the identification of the etiology of abnormal and normal bone cell activity and the assessment of this activity, is the desire and need to develop compositions and methods for the treatment of abnormal bone cell activity, as a result of disease, surgical removal or physiological trauma all of which lead to bone tissue loss. Therapeutics which provide for the replacement and repair of bone tissue, such as with the use of bone implants, are highly desired.
Several groups have attempted to provide compositions suitable for the therapeutic replacement of bone tissue. U.S. Pat. No. 4,871,578 discloses a process for the formation of a non-porous smooth coating of hydroxyapatite suitable for implant use. U.S. Pat. No. 4,983,182 discloses a ceramic implant which comprises a sintered body of zirconia and a coating of α-TCP and zirconia, or hydroxyapatite and zirconia. U.S. Pat. No. 4,988,362 discloses a composition for the fusion of a bioceramic to another bioceramic. U.S. Pat. No. 4,990,163 discloses a coating used for the production of bioceramics which consist of α-TCP and β-TCP. Although these different compositions may be used as biocompatible coatings for implants and the like, none of these compositions have been demonstrated to participate in the natural bone remodeling process. Furthermore, none of the prior compositions developed, can be manipulated to reliably produce a range of films, thicker coatings and bulk ceramic pieces which share a common composition and morphology which leads to similar bioactive performance in vivo and in vitro.
It has therefore long been the goal of biomaterials research in the field of orthopedics to develop synthetic structures exhibiting comprehensive bioactivity. Bioactive synthetic substrates and scaffolds capable of incorporation into the natural process of bone remodeling are of interest in applications which include not only in vitro bone cell assays (Davies, J., G. Shapiro and B. Lowenberg. Cells and Materials 3(3) 1993; pp. 245-56), but also in vivo resorbable bone cements (Gerhart, T., R. Miller, J. Kleshinski and W. Hayes. J Biomed Mater Res 22 1988; pp. 1071-82 and Kurashina, K., H. Kurita, M. Hirano, J. deBlieck, C. Klein and K. deGroot. Journal of Materials Science: Materials in Medicine 6 1995; pp. 340-7), implantable coatings which enhance the bonding of natural bone to the implant (Tofe, A., G. Brewster and M. Bowerman. Characterization and Performance of Calcium Phosphate Coatings for Implants edited by E. Horowitz and J. Parr. Philadelphia: ASTM, pp. 9-15 (1994), various forms of implantable prostheses and bone repair agents (Tolman, D. and W. Laney. Mayo Clin Proc 68 1993; pp. 323-31 and Levitt, S., P. Crayton, E. Monroe and R. Condrate. J Biomed Mater Res 3 1969; pp. 683-5), and ex vivo tissue engineering (Kadiyala,S., N. Jaiswal, S. Bruder. Tissue Engin 3(2) 1997; pp. 173-84). The prime objective for such materials in vivo is to combine the stimulation of osteogenic activity in associated bone tissues for optimum healing, with the capability to be progressively resorbed by osteoclasts during normal continuous remodeling (Conklin, J., C. Cotell, T. Barnett and D. Hansen. Mat Res Soc Symp Proc 414 1996; pp. 65-70). In vitro, related functions are to provide standardized laboratory test substrates on which osteoclast resorptive function or osteoblast production of mineralized bone matrix can be assessed and quantified (Davies, J., G. Shapiro and B. Lowenberg. Cells and Materials 3(3) 1993; pp. 245-56). Such substrates must be stable and insoluble in the biological environment until acted upon by osteoclasts, the specific bone mineral resorbing cells.
While calcium hydroxyapatite (Ca5(OH)(PO4)3 or HA) is the primary inorganic component of natural bone (Yamashita, K., T. Arashi, K. Kitagaki, S. Yamada and T. Umegaki. J Am Ceram Soc 77 1994; pp. 2401-7), trace elements are also present (Biominerals edited by F. Driessens and R. Verbeeck. Boston: CRC Press (1990). Calcium hydroxyapatite is but one of a number of calcium-phosphorous (Ca—P) compounds which are biocompatible. Others include octacalcium phosphate (Brown, W., M. Mathew and M. Tung. Prog Crys Growth Charact 4 1981; pp. 59-87) and both phases of tricalcium phosphate (Ca3(PO4)2 or α-TCP/β-TCP) (Elliott J. Structure and Chemistry of the Apatites and Other Calcium Orthophosphates New York: Elsevier (1994). Compounds, particularly HA, may show differing degrees of stoichiometry with the Ca/P ratio ranging from 1.55 to 2.2 (Meyer, J. and B. Fowler. Inorg Chem 21 1997; pp. 3029-35). Such materials can be artificially created by conventional high temperature ceramic processing (Santos, R. and R. Clayton. American Mineralogist 80 1995; pp. 336-44) or by low temperature aqueous chemistry (Brown, P., N. Hocker and S. Hoyle. J Am Ceram Soc 74(8) 1991; pp. 1848-54 and Brown, P. and M. Fulmer. J Am Ceram Soc 74(5) 1991; pp. 934-40). Most of such artificial materials show good biocompatibility in that bone cells tolerate their presence with few deleterious effects, and indeed enhanced bone deposition may occur (Ito, K., Y. Ooi. CRC Handbook of Bioactive Ceramics edited by T. Yamamuro, L. Hench and J. Wilson. Boca Raton, Fla.: CRC Press, pp. 39-51 (1990) and Ohgushi, H., M. Okumura, S. Tamai, E. Shors and A. Caplan. J Biomed Mater Res 24 1990; pp. 1563-70). Currently, the most recognized medical application of calcium phosphates is the coating of implantable prosthetic devices and components by thermal or plasma spray to render the surface osteoconductive. It has been noted that Ca—P ceramics which are stable in biological environments are often a mixture of individual compounds (LeGeros, R., G. Daculsi. CRC Handbook of Bioactive Ceramics edited by T. Yamamuro, L. Hench and J. Wilson. Boca Raton: CRC Press, (1990)). However, despite the osteogenic potential of these artificial materials, none actively participate in the full process of natural bone remodeling.
In an effort to understand the cellular mechanisms involved in the remodeling process, several research groups have attempted to develop methods to directly observe the activity of isolated osteoclasts in vitro. Osteoclasts, isolated from bone marrow cell populations, have been cultured on thin slices of natural materials such as sperm whale dentine (Boyde et al Brit. Dent. J. 156, 216, 1984) or bone (Chambers et al J. Cell Sci. 66, 383, 1984). The latter group have been able to show that this resorptive activity is not possessed by other cells of the mononuclear phagocyte series (Chambers & Horton, Calcif Tissue Int. 36, 556, 1984). More recent attempts to use other cell culture techniques to study osteoclast lineage have still had to rely on the use of cortical bone slices (Amano et al. and Kerby et al J. Bone & Min. Res. 7(3)) for which the quantitation of resorptive activity relies upon either two dimensional analysis of resorption pit areas of variable depth or stereo mapping of the resorption volume. Such techniques provide at best an accuracy of approximately 50% when assessing resorption of relatively thick substrata. In addition these analysis techniques are also very time consuming and require highly specialized equipment and training. Furthermore, the preparation and subsequent examination of bone or dentine slices is neither an easy nor practical method for the assessment of osteoclast activity.
The use of artificial calcium phosphate preparations as substrata for osteoclast cultures has also met with little success. Jones et al (Anat. Embryol 170, 247, 1984) reported that osteoclasts resorb synthetic apatites in vitro but failed to provide experimental evidence to support this observation. Shimizu et al (Bone and Mineral 6, 261, 1989) have reported that isolated osteoclasts resorb only devitalized bone surfaces and not synthetic calcium hydroxyapatite. These results would indicate that functional osteoclasts are difficult to culture in vitro.
In the applicant's published international PCT application WO94/26872, cell-mediated resorption was shown to occur on a calcium phosphate-based thin film formed by the high temperature processing of a calcium phosphate colloidal suspension on quartz substrates. When used in vitro, these ceramic films exhibited multiple discrete resorption events (lacunae) across their surface as a result of osteoclast activity, with no evidence of dissolution arising from the culture medium. The regular margins of these lacunae correspond closely to the size and shape of the ruffled borders normally produced by osteoclasts as the means by which they maintain the localized low pH required to naturally resorb bone mineral in vivo. Enhanced deposition of mineralized bone matrix also occurs on these ceramics in the presence of osteoblasts.
It is now demonstrated by the Applicant's that these thin film ceramics exhibit two general characteristics: (1) the presence of a mixture of Ca—P containing phases comprising approximately 33% HA and approximately 67% of a silicon stabilized calcium phosphate and (2) a unique morphology. Importantly, it was noted that the thermal processing of the Ca—P colloid at 1000° C. resulted in an HA powder, while the same colloidal suspension processed on quartz had a mixed HA and silicon stabilized calcium phosphate phase composition. Energy dispersive X-ray analysis of the film demonstrated the presence of Si in the coating while cross-sectional transmission electron microscopy indicated a microporous physical structure.
Applicants have discovered that the presence of stabilizing entities can stabilize the composition and prevent its degradation in physiological fluids. Hence, disappearance of calcium phosphate entities from a film, coating or bulk ceramic piece of this composition, is substantially due to the activity of the osteoclasts and not due to a dissolution process. The stabilized artificial bioactive composition is the first such composition which supports both osteoclast and osteoblast activity and which allows for the reliable assessment of the physiological activities of both cell types as well as for the development of both diagnostic and therapeutic strategies.
In view of the clinical importance of developing a synthetic bone graft that is both osteogenic and can participate in the body's natural cell-based remodeling process, it was important to focus on the role of introduced additives such as silicon in the formation of a calcium phosphate-based biomaterial compound capable of being assimilated and remodeled into natural bone with the aid of the activity of osteoclasts and osteoblasts. Since the compound could only be characterized by the preparation method, it was crucial to be able to both physically and chemically characterize the compound. In particular, it was important to characterize the physical structure of the compound and more importantly, the specific molecular and chemical structure of the stabilized compound in order to be able to understand why the new compound worked so well in biological conditions affecting the skeleton. The physical, molecular and chemical characterization of the compound could also provide for the development of further uses of the compound in the treatment of several different types of bone-related clinical conditions. In addition, this would also allow further chemical alteration of the compound in order that it could be designed for use in specific in vivo, in vitro and ex vivo applications.
The Applicant's work now pointed to the transformation of HA into a stabilized calcium phosphate phase. Surprisingly, during the difficult course of explicit characterization of the compound from a molecular standpoint, it was found-that the resultant stabilized compound was an entirely new compound herein described and termed Skelite™.